Monoclonal antibodies, hybridomas, improved method for determining the protein PTX3 and kit for said determination

ABSTRACT

Method for determining the level of PTX3 protein in a sample of a biological fluid; hybridoma capable of producing a rat anti-PTX3 monoclonal antibody where said hybridoma is selected from the group comprising MNB10 and Pen-3; specific anti-PTX3 rat monoclonal antibody selected from the monoclonal antibodies produced by the hybridomas MNB10 and Pen-3; kit for determining the level of PTX3 protein in a biological fluid wherein the said kit includes a rat anti-PTX3 monoclonal antibody.

The present invention relates to rat anti-PTX3 monoclonal antibodies,hybridomas for producing said antibodies, an improved method fordetermining the protein PTX3 in a biological fluid and a kit forperforming said determination.

More particularly, said method, said rat anti-PTX3 monoclonal antibodiesand said kit are useful for the early diagnosis of the risk of death inhuman individuals suffering from cardiovascular and/or cerebrovasculardiseases.

The pentraxins, so called on account of their pentameric structure, area group of proteins which include C-reactive protein (CRP) and serumamyloid P (SAP), produced by the liver in response to inflammatorymediators. The levels thereof in the serum increase in response tovarious stimuli and have been used for monitoring infections,inflammatory conditions and tissue damage.

PTX3 is a new member of this family which was found in endothelial cellsstimulated by interleukin-1 (IL-1). PTX3, a typical long-chainpentraxin, is characterized by a C-terminal region of 203 amino acidswhich displays homology with the classical pentraxins and by anN-terminal region of 178 amino acids devoid of homology. In contrast toCRP and SAP, PTX3 is produced in various types of cell, principally inendothelial cells and in mononuclear phagocytes, in response to IL-1 andto tumour necrosis factor (TNF), but not to interleukin-6 (IL-6),Further, PTX3 is produced by monocytes in response to components ofmycobacterial cell walls and by unstimulated synoviocytes in patientswith rheumatoid arthritis.

The protein PTX3 was identified as far back as 1992 (Breviario F. et al.“Cloning of a new gene related to C-reactive protein and serum amyloid Pcomponent” J. Biol. Chem. 1992, 267: 22190-22197).

It is also known that its production in bacteria was described by VidalAlles V. et al. in “Inducible expression of PTX3, a new member of thepentraxin family, in human mononuclear phagocytes.” Blood 1994, 84:3483-3493, while its production in eukaryotic cells (CHO) has beendescribed by Bottazzi B. et al. “Multimer formation and ligandrecognition by the long pentraxin PTX3—Similarities and differences withthe short pentraxin C-reactive protein and serum amyloid component.” J.Biol. Chem. 1997, 272: 32817-32823.

Nonetheless, the biological function of the protein PTX3 has not yetbeen fully understood.

Recent studies have demonstrated that the levels of PTX3 protein wereincreased in patients suffering from acute or chronic inflammatorydiseases such as sepsis or myocardial infarction. In particular, Peri etal. have reported that the levels of PTX3 protein reach peaks of6.94±11.26 ng/ml in infarcted patients 7.5 hours after admission tohospital coronary units (Circulation 2000, 102: 636-641).

Further, still more recently, it has been observed that the levels ofPTX3 protein in infarcted patients are indicative of the risk of deathin the three months following the episode (Latini R. et al., “Prognosticsignificance of the long pentraxin PTX3 in acute myocardial infarction:comparison with C-reactive protein, NT-proBNP and troponin T.” abstract3091, Supplement IV, page 680, Circulation 2003, 108 (17),). Moreparticularly, the authors reported that in a representative number ofpatients with myocardial infarction with elevation of the ST segment thelevels of PTX3 protein in the acute phase provide independentinformation predictive of the risk of death. The same prediction cannotbe made on the basis of the levels of C-reactive protein (short chainpentraxin) or of other biocardiac markers such as NT-proBNP ortroponin-T.

Further, the inventors are aware of experimental data which show that inpatients suffering from cerebral stroke, the level of PTX3 protein isproportional to the damage suffered by the central nervous system.

The determinations of PTX3 reported in the literature [Peri et al., loc.cit.; Muller B. et al., “Circulating levels of the long pentraxin PTX3correlate with severity of infection in critically ill patients” Crit.Care Med. 2001, 29(7): 1404-1407; Fazzini F. et al., “PTX3 insmall-vessel vasculitides—An independent indicator of disease activityproduced at sites of inflammation” Arthritis and Rheumatism 2001,44(12): 2841-2850] were performed by an ELISA method based on amonoclonal antibody specific for PTX3 protein and on a biotinylatedpolyclonal rabbit IgG specific for PTX3 protein. The aforesaidmonoclonal antibody is identified in the literature as MNB4 but thecorresponding hybridoma is not accessible to the public.

In particular, the aforesaid method is described in detail by Muller B.et al., loc. cit., and comprises the following steps:

a) 96-well ELISA plates (Nunc Roskilde, Denmark) were coated with 100 μlof rat monoclonal antibody MNB4 (as ascites, diluted 1:5000 in bufferused for the coating) and incubated for one night at 4° C.;

b) the plates were then thoroughly washed with a Dulbecco phosphatebuffer saline containing 0.05% Tween 20 (washing buffer) and 200 μl of5% milk powder to block non -specific binding sites;

c) after incubation for 2 hours at ambient temperature, the plates wereagain washed 3 times with washing buffer;

d) 50 μl of standard recombinant human PTX3 (from 100 pg/ml to 10 ng/ml)diluted in RPMI 1640 medium (Seromed, Berlin, Germany) and 2% bovineserum albumin (Sigma Chemicals, St. Louis, Mo.) or samples of testplasma, in triplicate, were placed in each well, and the plates wereincubated for 2 hours at 37° C.;

e) the plates were washed 3 times with washing buffer and 100 μl ofanti-TPX3 rabbit serum, conjugated with biotin, diluted 1:2000 inwashing buffer, were added;

f) the plates were incubated for 1 hour at 37° C. and then washed 3times with 200 μl of washing buffer;

g) 100 μl of streptavidin-peroxidase conjugated with dextran substrate(Amdex, Copenhagen, Denmark), diluted 1:4000, were added to each well,and the plates were incubated for 1 hour at ambient temperature;

h) after the plates had been washed 4 times, 100 μl of the chromogenicsubstrate ABTS Microwell Peroxidase Substrate System (Kirkegaard andPerry Laboratories, Gaithersburg, Md.) were added;

i) the plates were read at 405 nm with an automatic reader.

One aspect of the present invention is based on the fact that theinventors have observed that the aforesaid known method presents anumber of disadvantages.

A first disadvantage of the known method consists in the fact that, inthe case of the plasma of some patients, the levels of PTX3 determinedat different dilutions of the test plasma sample are not proportional tothe dilution performed. The inventors have now found that thisdisadvantage is surprisingly overcome by adding EDTA to the test plasmasample. In spite of the fact that the reason for this has not yet beenentirely elucidated, the inventors postulated, without desiring therebyto limit the scope of the invention, that the observed effect is due tothe ability of EDTA to complex Mg⁺⁺ and Ca⁺⁺ ions, so that the sameresult should also be obtained with other complexing agents.

A second disadvantage consists in the fact that the sensitivity of theknown method (ca. 200 pg/ml) is not sufficient for determining PTX3protein in about 5% of normal subjects. The inventors have now foundthat the sensitivity of the method can be increased to 75 pg/ml by usingnovel monoclonal antibodies (step a), changing the concentration ofstreptavidin-peroxidase (step g) and using a different chromogen (steph).

In one aspect thereof, the invention thus relates to a method fordetermining the level of PTX3 protein in samples of a biological fluidcomprising the following steps:

-   i) 96-well ELISA plates are coated with 100 μl of a solution    containing a rat monoclonal antibody and incubated for one night at    4° C.;-   ii) the plates are then washed with a buffer solution and a solution    capable of blocking the non-specific binding sites;-   iii) after incubation for 2 hours at ambient temperature, the plates    are again washed with washing buffer;-   iv) in duplicate, 50 μl of standard recombinant human PTX3 diluted    in a suitable medium or samples of the biological fluid under test    are placed in each well and the plates are incubated for 2 hours at    37° C.;-   v) the plates are washed repeatedly with washing buffer and 100 μl    of 25 ng/ml biotinylated anti-PTX3 rabbit IgG in washing buffer are    then added to each well;-   vi) the plates are incubated for 1 hour at 37° C. and then washed    repeatedly with washing buffer;-   vii) 100 μl of diluted streptavidin-peroxidase are added to each    well, and the plates are incubated for 1 hour at ambient    temperature;-   viii) after the plates have been washed repeatedly with washing    buffer, 100 μl of chromogenic substrate are added to each well;-   ix) the plates are briefly incubated at ambient temperature, a stop    solution is added and the plates are read at 405 nm with an    automatic reader;    wherein the improvement consists in the fact that:    -   the rat monoclonal antibody used in step (i) is the antibody        obtained from the hybridoma MNB10 (access No. ABC/PD04001) or        from the hybridoma Pen-3 (access No. ABC/PD01004);    -   the streptavidin-peroxidase used in step (vii) is diluted        1:8000; and    -   the chromogenic substrate used in step (viii) is        tetramethylbenzidine (TMB).

Preferably, in step (i) the concentration of rat monoclonal antibody inthe solution is about 700 ng/ml. Further, the solution used in step (i)advantageously consists of a coating buffer solution. Preferably thesaid buffer solution contains 15 mM of carbonate buffer and its pH is9.6.

Typically, the buffer solution used in step (ii) consists of PBS(phosphate buffer saline)+0.05% of Tween 20. Advantageously, the saidsolution is used in the amount of about 300 μl/well.

Preferably, the solution capable of blocking non-specific binding sitesused in step (ii) consists of a 5% solution of milk powder in a buffersolution consisting of PBS+0.05% of Tween 20. Advantageously, the saidsolution capable of blocking non-specific binding sites is used in theamount of about 300 μl/well.

The washing specified in step (iii) is preferably repeated 3 times, eachtime using about 300 μl of solution for each well.

Preferably, in step (iv) the standard recombinant human PTX3 used isplaced in the wells in quantities increasing from 75 pg/ml to 1.2 ng.Advantageously, the medium used to dilute the test plasma samplesconsists of PBS+2% of BSA (bovine serum albumin)+0.18% of K₃-EDTA. Thepresence of EDTA in this solution is very important since, as alreadystated, the inventors have found that this makes it possible to obtainPTX3 measurements proportional to the dilution performed.

The biotinylated anti-TPX3 rabbit IgG used in step (v) is preferablyobtained according Muller B. et al., loc. cit.

Also, the streptavidin-peroxidase used in step (vii) is preferably thehorseradish peroxidase-conjugated streptavidin Amdex (RPN 4401 Amersham,Copenhagen, Denmark).

Typically, the stop solution used in step (ix) is a 1M solution ofH₂SO₄.

Advantageously, the TMB substrate solution used in step (xii)corresponds to the catalogue number 2642 KK of the firm Pharmingen.

In another aspect, the present invention relates to hybridomas capableof producing an anti-PTX3 rat monoclonal antibody and designated MNB 10and Pen-3. MNB 10 was deposited on Apr. 16, 2004 under the terms of theBudapest Treaty and is identified by Access Number ABC/PD04001 at theAdvanced Biotechnology Centre and Pen-3 was deposited on Feb. 8, 2001under the terms of the Budapest Treaty and is identified as AccessNumber ABC/PD01004. The full address of the depository is:

-   -   Advanced Biotechnology Center (ABC)    -   Interlab Cell Line Collection    -   (Biotechnology Dept.)    -   Largo Rossana Benzi, 10    -   16132 Genova    -   Italy.

The selection of the aforesaid hybridomas which produce anti-PTX3 ratmonoclonal antibodies according to the invention can be carried out byconventional methods such as those for example described in Example 1below.

In another aspect, the present invention relates to a specific anti-PTX3rat monoclonal antibody selected from the group comprising themonoclonal antibodies produced by the aforesaid hybridomas MNB10 andPen-3.

The preparation of the specific anti-PTX3 rat monoclonal antibodies fromthe hybridomas of the present invention is not subject to particularrestrictions and can be carried out by conventional methods such asthose for example described in Example 2 below.

In yet another aspect, the present invention relates to a kit for thedetermination of the level of PTX3 protein in biological fluids,characterized in that it includes an anti-PTX3 rat monoclonal antibody.

In a preferred embodiment, the determination of the level of PTX3protein is carried out on the serum of a human individual suffering froma cardiovascular and/or cerebrovascular disease for early diagnosis oftheir risk of death.

Preferably, the aforesaid kit also includes an anti-PTX3 rabbitpolyclonal antibody.

Advantageously, the aforesaid kit also includes purified recombinantPTX3 protein.

In a preferred embodiment, the aforesaid kit also includes streptavidinconjugated with horseradish peroxidase.

Advantageously, the aforesaid kit also includes a washing buffersolution.

Preferably, the aforesaid kit also includes a diluent for the samples ofbiological fluid to be assayed.

Typically, the aforesaid kit also includes, as chromogen, a solution oftetramethylbenzidine (TMB).

Finally, a stop solution can also be included in the aforesaid kit.

FIG. 1 below and the examples that follow serve further to illustratethe invention, without however limiting it.

FIG. 1 is a diagram which illustrates the effect of EDTA on the plasmaof a patient with cardiac decompensation.

In FIG. 1, the dilutions of whole plasma (1:2, 1:4, 1:8 and 1:16) areshown on the x-axis. The optical density is shown on the y-axis.

As can be seen, the levels of PTX3 protein detectable in the serum of apatient with cardiac decompensation in the whole plasma dilutioninterval between about 1:4 (0.25) and about 1:16 (0.0625) are, in theabsence of EDTA, about three times lower than the levels of proteinactually present. However, the addition of EDTA makes it possible toobtain levels that essentially correspond to those of the standardcurve.

EXAMPLE 1 Hybridoma

Immunisation

Lewis rats are subcutaneously immunised with 200 μg of PTX3 three timesat intervals of 15 days. After evaluation of the antibody response by anELISA assay, the fusion is performed with the animals with the highestantibody titer.

Fusion Protocol

Method

a) Preparation of the Splenocytes

-   -   the spleen of the previously immunised rat is removed under        sterile conditions and washed 3 times with 10 ml of DMEM        (Dulbecco's modified Eagle's medium), transferred into a plastic        Petri dish containing 3 ml of DMEM medium and disaggregated by        means of needles and/or crushed with a syringe piston;    -   the cell suspension is transferred to a test tube, made up to 50        ml with DMEM medium and filtered with a 70 μm screen so as to        remove cell aggregates;    -   wash the splenocytes twice with 50 ml of DMEM medium;    -   count in Turk.

b) Preparation of the Myeloma “SP2/0”

-   -   the myeloma must be in exponential growth phase and not plateau.    -   wash the cells twice with 50 ml of DMEM medium.    -   count them

c) Fusion

-   -   add the cells of SP2/0 to the splenocytes so as to obtain a        ratio of (5:1) between splenocytes and myeloma;    -   make up to 50 ml with DMEM and centrifuge at 1700 rpm for 7 min;    -   draw off the supernatant with a Pasteur pipette taking care to        remove it all;    -   stir the cells that have sedimented, shaking the test tube with        the finger and then add 0.6 ml of 37% PEG in DMEM maintained at        37° C.;    -   with a Pasteur pipette, very slowly resuspend the cells, wait 2        min from when the PEG was added, and then centrifuge at 800 rpm        for 6 min;    -   remove the supernatant with a Pasteur pipette. Resuspend the        cells, shaking the test tube gently with the finger and add DMEM        maintained at 37° C. drop by drop, up to 20 ml volume;    -   centrifuge at 1300 rpm for 10 min;    -   remove the supernatant, and resuspend the pellet of cells very        gently with HAT-DMEM using a Pasteur pipette;    -   dilute them to a concentration of 1.25×10⁶×ml and then seed 0.2        ml of them per well in flat-bottomed 96-well plates, so as to        have 2.5×10⁵ cells per well;    -   incubate the plates at 37° C. in a well humidified incubator and        check the presence of colonies after one week;    -   7 days after the fusion, draw off the medium and add 200 μl of        fresh HAT-DMEM;    -   perform the screening between the 10^(th) and 15^(th) days after        the fusion;    -   then clone the positive hybridomas in HT-DMEM medium;    -   after the second cloning, HT-DMEM medium can be replaced with        DMEM for hybridomas.

Materials

DMEM medium (1x) DMEM 500 ml L-glutamine 5 ml gentamicin 0.5 ml Mediumfor hybridomas (DMEM) (1x) DMEM 500 ml *FCS (fetal calf serum) 50 mlnon-essential AAs (Amino Acids) 5 ml sodium pyruvate 5 ml L-glutamine 5ml gentamicin 0.5 ml HAT-DMEM Myeloma medium + hypoxanthine +aminopterin + thymidine HT-DMEM Myeloma medium + hypoxanthine +thymidine PEG 1550 (polyethylene glycol 1550) 37%

Autoclave 7.4 g of PEG (Serva code 33132) in a pyrex bottle. Before itsolidifies (ca. 55° C.) add 12.6 ml of DMEM without FCS and mix well.Filter with a 0.2 μm filter, aliquot 1 ml per test tube and store inrefrigerator.

EXAMPLE 2 Purification of Monoclonal Antibodies Using Sepharose-BoundProtein G Materials

-   -   PBS with Ca and Mg: 450 ml of distilled water+50 ml of PBS        (10×)+3 ml of 1M NaOH;    -   Sepharose Protein G 4 fast flow (Pharmacia cat. 17-0618-01):        wash the resin 4 times with PBS by decantation or light        centrifugations, then dilute it to 10% with PBS+0.1% of sodium        azide and store it in the refrigerator;    -   glycine—0.1M HCl buffer pH 2.8: dissolve 750 mg of glycine in        100 ml of distilled water and adjust to pH 2.8 with 280 μl of        37% HCl;    -   1.5M TRIS-HCl buffer pH 8.8: dissolve 18.17 g of TRIS in 100 ml        of distilled water and adjust to pH 8.8 with 37% HCl;    -   One 7 ml plastic minicolumn.

Method

-   -   load the column to 3 ml volume with the resin Sepharose Protein        G 4 fast flow;    -   wash with 20 ml of PBS taking care not to let the resin dry out.    -   dilute the ascites or the serum 1:3 with PBS, and filter on a        0.2 μm filter;    -   pass through the resins 4 times and at the end stop the flow at        the level of the resin;    -   wash with 30 ml of PBS;    -   add 9 ml of pH 2.8 glycine—HCl buffer (3 ml at a time) and        collect the eluate in 3 test tubes;    -   adjust immediately to pH 7 by adding from 100 to 150 μl of pH        8.8 TRIS-HCl buffer;    -   measure the total proteins and if necessary concentrate them        with Centriplus 50 or 100;    -   dialyse against PBS, aliquot and freeze.

EXAMPLE 3 Method for Determining the Levels of PTX3 in Patients' Plasma

-   -   96-well ELISA plates (Nunc MaxiSorp 446612) were coated with 100        μl of a coating buffer solution (15 mM carbonate buffer, pH 9.6)        containing purified MNB10 antibody (700 ng/ml) and incubated for        one night at 4° C.;    -   the plates were washed 3 times with 300 μl/well of a washing        buffer solution (PBS+0.05% Tween 20) and then 300 μl of washing        buffer with 5% of milk powder were added to block non-specific        binding sites;    -   after incubation for 2 hours at ambient temperature, the plates        were washed 3 times with washing buffer;    -   in duplicate, 50 μl of standard recombinant human PTX3 (from 75        pg/ml to 1.2 ng/ml) and samples of the plasma under test diluted        in PBS+2% BSA+0.19% K₃-EDTA were placed in each well and the        plates were incubated for 2 hours at 37° C.    -   the plates were washed 5 times with washing buffer and 100 μl of        25 ng/ml biotinylated rabbit anti-PTX3 IgG in washing buffer        were added to each well;    -   the plates were incubated for 1 hour at 37° C. and then washed 5        times with 300 μl of washing buffer;    -   100 μl of horseradish peroxidase-conjugated streptavidin Amdex        (RPN 4401 Amersham Copenhagen, Denmark) diluted 1:8000 were        added to each well and the plates were incubated for 1 hour at        ambient temperature;    -   after the plates had been washed 5 times with washing buffer,        100 μl of a substrate solution of TMB (tetramethylbenzidine)        were added to each well    -   the plates were incubated for 5 minutes at ambient temperature;    -   50 μl of stop solution (H₂SO₄, 1M) were added to each well;    -   the absorbance at 405 nm was read within 30 minutes from the        stopping of the reaction.

EXAMPLE 4 Kit

A kit for determining the levels of PTX3 in human biological fluidscomprises:

-   1. Microplate: 12×8 wells coated with rat anti-PTX3 antibody MNB10.-   2. Biotinylated rabbit anti-PTX3 IgG in phosphate buffer solution.-   3. Horseradish peroxidase-conjugated streptavidin in phosphate    buffer solution.-   4. Standards: purified recombinant PTX3 at 2.4, 1, 0.5, 0.2 and 0.05    ng/ml in buffer solution.-   5. Washing buffer solution: phosphate buffer saline (PBS) solution.-   6. Diluent (to dilute the human biological fluid under test): 1%    bovine serum albumin and 0.19% K3-EDTA in phosphate buffer saline    solution.-   7. Substrate: 0.26 mg/ml tetramethylbenzidine and 0.01% H₂O₂    stabilised in 0.05 mol/l citrate buffer (pH 3.8).-   8. Stop solution: 1 M H₂SO₄.

1. Hybridoma cell line MNB10 identified by Access No. ABC/PD04001 at theAdvanced Biotechnology Center of Genoa, Italy.
 2. Monoclonal antibodyMNB10 produced by the hybridoma cell line MNB10 deposited at theAdvanced Biotechnology Center of Genoa, Italy under Access No.ABC/PD04001.
 3. A kit comprising the monoclonal antibody MNB10 of claim2 and a microplate or microtiter plate.
 4. A kit comprising themonoclonal antibody MNB10 of claim 2 and one or more ingredientsselected from the group consisting of a biotinylated rabbit anti-PTX3IgG, horseradish peroxidase-conjugated streptavidin, purified longpentraxin PTX3, phosphate buffer solution, washing buffer solution,diluent for a biological sample suspected of containing PTX3,chromogenic substrate, and stop solution.
 5. A method for detecting longpentraxin PTX3 in a liquid biological sample comprising: contacting aliquid biological sample with the monoclonal antibody MNB10 of claim 2,and detecting a presence or an amount of PTX3 in the liquid biologicalsample based on a presence or an amount of complex formation betweensaid monoclonal antibody and PTX3 in the liquid biological sample. 6.The method of claim 5 which is an Enzyme-Linked Immunosorbent Assay(ELISA) and wherein said monoclonal antibody MNB10 is bound to amicroplate or microtiter plate.
 7. The method of claim 6, wherein saidELISA comprises a step of binding biotinylated rabbit anti-PTX3 IgG toPTX3 bound to said monoclonal antibody MNB10.
 8. The method of claim 7,wherein said ELISA comprises a step of binding streptavidin conjugatedwith horseradish peroxidase to the biotinylated IgG antibody.
 9. Themethod of claim 8, wherein said ELISA employs tetramethylbenzidine (TMB)as a chromogenic substrate.
 10. The method of claim 6, wherein saidELISA comprises: binding monoclonal antibody MNB10 to microtiter platewells, blocking unbound sites and removing unbound material by washing,applying the liquid biological sample to which EDTA has been added tosaid microtiter plate wells under conditions suitable for binding ofPTX3 in the sample to the MNB10 monoclonal antibody in said wells,washing to remove unbound material, applying a biotinylated anti-PTX3IgG to said microtiter plate wells under conditions suitable for bindingof the IgG to PTX3 bound in said wells, washing to remove unboundmaterial, applying streptavidin-peroxidase to said wells underconditions suitable for binding to the biotinylated IgG antibodies boundin said wells, washing to remove unbound material, and adding achromogenic substrate comprising tetramethylbenzidine (TMB). 11.Hybridoma cell line Pen-3 identified by Access No. PD01004 at theAdvanced Biotechnology Center of Genoa, Italy.
 12. Monoclonal antibodyPen-3 produced by hybridoma cell line Pen-3 identified by Access No.PD01004 at the Advanced Biotechnology Center of Genoa, Italy.
 13. A kitcomprising the monoclonal antibody Pen-3 of claim 12 and a microplate ormicrotiter plate.
 14. A kit comprising the monoclonal antibody Pen-3 ofclaim 12 and one or more ingredients selected from the group consistingof a biotinylated rabbit anti-PTX3 IgG, horseradishperoxidase-conjugated streptavidin, purified PTX3, phosphate buffersolution, washing buffer solution, diluent for a biological samplesuspected of containing long pentraxin PTX3, chromogenic substrate, andstop solution.
 15. A method for detecting long pentraxin PTX3 in aliquid biological sample comprising: contacting a liquid biologicalsample with the monoclonal antibody Pen-3 of claim 12, and detecting apresence or an amount of PTX3 in the liquid biological sample based on apresence or an amount of complex formation between said monoclonalantibody and PTX3 in the liquid biological sample.
 16. The method ofclaim 15 which is an Enzyme-Linked Immunosorbent Assay (ELISA) andwherein said monoclonal antibody is bound to a microplate or microtiterplate.
 17. The method of claim 16, wherein said ELISA comprises a stepof binding biotinylated rabbit anti-PTX3 IgG to PTX3 bound to saidmonoclonal antibody Pen-3.
 18. The method of claim 17, wherein saidELISA comprises a step of binding streptavidin conjugated withhorseradish peroxidase to the biotinylated IgG antibody.
 19. The methodof claim 18, wherein said ELISA employs tetramethylbenzidine (TMB) as achromogenic substrate.
 20. The method of claim 16, wherein said ELISAcomprises: binding monoclonal antibody Pen-3 to microtiter plate wells,blocking unbound sites and removing unbound material by washing,applying the liquid biological sample to which EDTA has been added tosaid microtiter plate wells under conditions suitable for binding ofPTX3 in the sample to the PEN-3 monoclonal antibody in said wells,washing to remove unbound material, applying a biotinylated anti-PTX3IgG to said microtiter plate wells under conditions suitable for bindingof the IgG to PTX3 bound in the wells, washing to remove unboundmaterial, applying streptavidin-peroxidase to said wells underconditions suitable for binding to the biotinylated IgG antibodies boundin said wells, washing to remove unbound material, and adding achromogenic substrate comprising tetramethylbenzidine (TMB).